Simultaneous Estimation of Lamotrigine and Clozapine by Simultaneous equation method in their Synthetic Mixture which use in Schizophrenia

 

Priyanka P. Atodariya*, Hasumati A. Raj, Vineet C. Jain

Shree Dhanvantary Pharmacy Collage Kim, Surat, Gujarat, India

*Corresponding Author E-mail: atodariya.priyanka@yahoo.com, drharaj@yahoo.com

 

ABSTRACT:

Two simple spectroscopic methods have been developed for simultaneous estimation of Lamotrigine and Clozapine in synthetic mixture. Simultaneous equation method involves the measurement of absorption at two wavelengths 308 nm (λmax for Lamotrigine) and 259.60 nm (λmax for Clozapine). The method was found linear between the range of 1-5 g/ml for Lamotrigine and 6-30 g/ml for Clozapine for method .The accuracy and precision was determined and validated statistically. Both the method showed good reproducibility and recovery with %RSD less than 1.The method was found to be rapid, specific, precise and accurate and can be successfully applied for the routine analysis for Lamotrigine and Clozapine in bulk and combined dosage form.

 

KEYWORDS: Lamotrigine, Clozapine, Simultaneous equation Method.

 

 


INTRODUCTION:

Lamotrigine is an epileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters[1]. IUPAC name of Lamotrigine is 3, 5-diamino-6-2, 3(dichlorophenyl)-1, 2, 4 triazine[2].Clozapine is benzodiazepine derivative and use in treatment of schizophrenia[3]. The IUPAC name of Clozapine is 8-Chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo [b, e] [1, 4] diazepine [4]. It acts by inhibiting presyneptic voltage sensitive sodium channels and excitatory neurotransmitter release . Dopamine is one of the important neurotransmitter and plays a significant role in the functioning of central nervous system [5]. The inhibition of dopamine transmission through blockade of dopamine D2 receptors is considered to be essential for antipsychotic efficacy, but it is postulated that modulation of glutamate transmission may be equally important. In support of this, symptoms similar to schizophrenia can be induced in healthy volunteers using N-methyl-D-aspartate (NMDA) antagonist drugs that are also known to enhance glutamate transmission.[6]

 

Furthermore, Lamotrigine, which can modulate glutamate release, may add to or synergise with atypical antipsychotic drugs, some of which may themselves modulate glutamate transmission [7].So, Lamotrigine use with Clozapine in treatment of schizophrenia.

 

Chemical structure of Lamotrigine and Clozapine

 

Fig-1 Lamotrigine[8]

 

Fig-2 Clozapine[9]

 

MATERIAL AND METHODS:

Apparatus and instrument:

A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was used to measure absorbance of all the solutions.

Spectra were automatically obtained by UV-Probe system software.

An analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing the samples.

Sonicator (D120/2H, TRANS-O-SONIC)

Class B volumetric glassware were used (Borosilicate)

All instruments and glass wares were calibrated.

 

Reagents and material:

Lamotrigine raw material was received as gift sample from Praveen Laboratories.

Clozapine raw material was received as gift sample from ZCl Pharmaceuticals Pvt. Ltd.

Methanol AR Grade (Rankem), Distilled Water, 0.1 N HCl, 0.1 N NaOH were used for development purpose.

 

For simultaneous equation method preparation of standard solution:

Standard solution of Lamotrigine (LAMO):

Preparation of stock solution of LAMO:

Accurately weighed quantity of LAMO 10 mg was transferred to 100ml volumetric flask, dissolved, and diluted up to mark with methanol to give a stock solution having strength 100g/ml.

 

Standard solution of Clozapine (CLO):

Preparation of stock solution of CLO:

Accurately weighed quantity of CLO 10 mg was transferred into 100 ml volumetric flask, dissolved and diluted up to mark with methanol to give a stock solution having strength 100g/ml.

 

Preparation of Standard Mixture Solution (LAMO + CLO):

1ml of standard stock solution of LAMO (100μg/ml) and 6 ml of standard stock solution of CLO (100μg/ml) was pipette out into two 10ml volumetric flasks and volume was adjusted to the mark with methanol to get 10μg/ml of LAMO and 60μg/ml of CLO.

 

Preparation of test solution:

The preparation of synthetic mixture was as per patent:

         Lamotrigine: 10mg

         Clozapine: 60mg

         Sodium Starch Glycolate: 10 mg

         Starch: 10 mg

         Magnesium stearate: 10 mg

 

All the excipients were mixed in 100ml volumetric flask then make up the volume with methanol and sonicated for 15min.after sonicate make up the volume up to 100 ml with methanol. The solution was filtered through Whatman filter paper No. 42. Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

 

Validation of proposed method:

Parameters to be considered for the validation of methods are:

1) Linearity and Range:

Procedure:

Calibration curves for Lamotrigine:

The linearity response was determined by analyzing 6 independent levels of calibration curve in the range of 1-5μg/ml OF LAMO .This series consisted of five concentrations of standard LAMO solution ranging from 1-5 μg /ml. The solutions were prepared by pipetting out Standard LAMO stock solution (0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order spectrum, measured the absorbance at 308 nm against a reagent blank solution (Methanol).

 

Calibration curve for Clozapine:

The linearity response was determined by analyzing 6 independent levels of calibration curve in the range of 6-30 μg /ml CLOZ. This series consisted of five concentrations of standard CLOZ solution ranging from 6-30 μg/ml. The solutions were prepared by pipetting out Standard CLOZ stock solution (0.6ml, 1.2ml, 1.8ml, 2.4ml, and 3.0ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order spectrum measured the absorbance at 259.50 nm against a reagent blank solution (Methanol).

 

2) Precision:

I. Intraday precision:

Procedure:

The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1,3,5μg/ml for LAMO and 6,18,30 μg/ml for CLO and three replicate (n=3)each on same day.

 

For zero order spectra was measured at 308 nm for LAMO and 259.50 nm for CLO.

 

The % RSD value of the results corresponding to the absorbance was expressed for intra-day precision.

 

II. Interday Precision:

Procedure:

The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1,3,5 μg/ml for LAMO and 6,18,30μg/ml for CLO and three replicate (n=3)each on different day.

 

For zero order spectra was measured at 308 nm for LAMO and 259.50 nm for CLO.

The % RSD value of the results corresponding to the absorbance was expressed for inter-day precision.

 

3) Accuracy:

It was determined by calculating the recovery of LAMO and CLO by standard addition method.

 

Accuracy was done by adding both API standard solution and test solution. Total concentration was as per table.1

 

Table 1: Solution for accuracy study

Concentration of Formulation (g/ml)

Concentration of API in spiking solution (g/ml)

Total concentration of (μg/ml)

LAMO

CLO

LAMO

CLO

LAMO

CLO

2

12

1.8

10

3.8

22

2

12

2

12

4

24

2

12

2.2

14

4.2

26

 

Procedure:

Each solution was taken and diluted with methanol up to 10ml volumetric flask and scanned between 200nm to 400nm against methanol as a blank. The amount of LAMO and CLO was calculated at each level and % recoveries were computed.

 

4) Limit of detection and quantitation:

The Limit of detection and quantitation of the developed method was assessed by analyzing ten replicates of standard solutions containing concentrations 1 μg/ml for LAMO and 6 μg/ml of CLO.

 

The LOD and LOQ were calculated as LOD = 3.3σ/S, and LOQ = 10σ/S, where σ is the standard deviation of the lowest standard concentration and S is the slope of the standard curve.% RSD was calculated.

 

5) Robustness and ruggedness:

Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the:

 

Change in Stock Solution Preparation,

 

Stock-1(10mg LAMO in 100ml methanol-100μg/ml and10mg CLO in 100ml methanol 100 μg/ml)

 

Stock-2(10mg LAMO in 50ml methanol-200μg/ml and 10mg CLO in 50ml methanol - 200 μg/ml)

 

Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450),

 

Three replicates were made for the concentration (1, 3, 5 μg/ml of LAMO and 6, 18, 30 μg/ml of CLO) with different stock solution preparation.

5) Analysis of EDA and ARG in synthetic mixture (assay):

Composition of synthetic mixture:

The preparation of synthetic mixture was as per patent:

Lamotrigine : 10mg

Clozapine :60mg

Sodium Starch Glycolate : 10 mg

Starch : 10 mg

Magnesium stearate : 10 mg

 

All the excipients were mixed in 100ml volumetric flask then make up the volume with methanol and sonicated for 15min.after sonicate make up the volume up to 100 ml with methanol. The solution was filtered through Whatman filter paper No. 42. Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

 

RESULT AND DISCUSSION:

Selection of wavelength and method development for determination of lamotrigine and clozapine

 

Fig -3 Overlain zero order spectra of Lamotrigine and Clozapine

 

The Simultaneous Equation Method Can be done as per Zero Order Spectra was overlay.

 

Spectra were taken in solvent Methanol in the range of 1-5 g/ml for LAMO and 6-30 g/ml for CLOZ.

 

Validation parameters:

1. Linearity and Range:

The Zero order spectra (fig.7.1) showed linear absorbance at 308 nm for LAMO (1-5 g/ml) and 259.50 nm for CLO (6-30 g/ml) with correlation coefficient (r2) of 0.998 and 0.998 for LAMO and CLO, respectively.

 

This method obeyed beers law in the concentration range 1-5 g/ml and 6-30g/ml for LAMO and CLO, respectively. (Table 7.2)

Correlation coefficient (r2) form calibration curve of LAMO and CLO was found to be 0.998and 0.998, respectively (figure 7.2)

 

The regression line equation for LAMO and CLO are as following,

 

y = 0.0362X+0.0044 for LAMO_____________ (1)

y = 0.0641 + 0.0294 for CLO _______________ (2)

 

Table 2:Calibration data for LAMO and CLO at 308 nm and 259.50 nm, respectively. *(n=6)

Lamotrigine Conc.

Absorbance SD*

Clozapine Conc.

Absorbance SD*

1

0.042 0.0012

6

0.436 0.0019

2

0.076 0.0012

12

0.796 0.0011

3

0.113 0.0006

18

1.155 0.0016

4

0.146 0.0016

24

1.548 0.0008

5

0.188 0.0021

30

1.984 0.0010

 

Fig-4 Calibration data for LAMO and CLO at 307 nm and 360 nm, respectively.*(n=6)

 

Table -3: Regression analysis data and validation parameter

Sr. No.

Statistical Parameter

Lamotrigine

Clozapine

1.

Concentration (g/ml)

1-5 g/ml

6-30 g/ml

2.

Straight line equation

Y=0.0362x+0.0044

Y=0.0641x+0.0294

Slope

0.0362

0.0641

Intercept

0.0044

0.0294

3.

Correlation coefficient (r2)

0.998

0.998

 

Mixture linearity [for simultaneous equation]:

 

Fig-5 Mixture Linearity in range of 1-5 g/ml of LAMO and 6-30 g/ml CLO (1:6 Ratio)

 

Table-4

Sr.no

Concentration in mixture

(LAMO:CLO) (1:6)g/ml

Absorbance

( 308 nm)

Absorbance

(259.50 nm)

1

1 : 6

0.231

0.521

2

2 : 12

0.432

0.916

3

3 : 18

0.642

1.446

4

4 : 24

0.844

1.801

5

5 : 30

1.027

2.243

 


Fig-6 Fig-7

 


2. Precision:

I. Intraday precision:

The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations 1 ,3, 5 μg/ml for LAMO and 6,18,30 μg/ml for CLO. Three replicate (n=3) each on same day for intraday.

II. Interday Precision:

The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations 1, 3, 5 μg/ml for LAMO and 6,18,30 μg/ml for CLO. Three replicate (n=3) each on different day for interday precision.

3. Accuracy:

Accuracy of the method was determined by recovery study from synthetic mixture at three levels (80%, 100%, and 120%) of standard addition.

The % recovery values are tabulated in Table 7.1.5 and 7.1.6

Percentage recovery for LAMO and CLO by this method was found in the range of 99.96 to 101.65%and 99.77-101.90%, respectively,

The value of %RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.


 

Table-5: Intraday precision data for estimation of LAMO and CLO *(n=3)

Precision

Conc.

LAMO (308 nm)

Conc.

CLO (259.50nm)

Intraday (n=3)

Abs. % RSD

1g/ml

0.232 0.53

6 g/ml

0.522 0.23

3 g/ml

0.642 0.26

18 g/ml

1.445 0.19

5 g/ml

1.025 0.25

30 g/ml

2.244 0.11

 

Table-6: Interday precision data for estimation of LAMO and CLO *(n=3)

Precision

Conc.

LAMO(308 nm)

Conc.

CLO(259.50nm)

Interday (n=3)

Abs. % RSD

1 g/ml

0.234 0.60

6 g/ml

0.535 0.84

3 g/ml

0.644 0.31

18 g/ml

1.456 0.28

5 g/ml

1.026 0.24

30 g/ml

2.244 0.12

 

Table 7: Recovery data of LAMO * and CLO* (n=3)

Initial

conc. (

g/ml)

Level

of recovery

Quantity of Std. Added

(g/ml)

Total

Amount

(g/ml)

Result of recovery study

Total Quantity Found*

(g/ml) %RSD

% recovery %RSD

LAM

CLO

 

LAM

CLO

LAM

CLO

LAM

CLO

LAM

CLO

2

12

0 %

-

-

2

12

2.030.81

12.100.38

101.970.18

100.850.30

2

12

80 %

1.8

10

3.8

22

3.820.12

22.210.03

100.840.23

100.910.38

2

12

100 %

2

12

4

24

4.050.20

24.440.14

101.110.44

101.730.12

2

12

120 %

2.2

14

4.2

26

4.230.44

26.260.09

100.540.28

100.980.20

Mean of 3 Determination

100.74%

101.91%

 

 


4. Limit of detection and quantitation

Table 8: LOD and LOQ data of LAMO and CLO*(n=10)

Parameter

LAMO*

CLO*

LOD (g/ml)

0.289

0.108

LOQ (g/ml)

0.877

0.329

 

5. Robustness and ruggedness:

Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the:

Change in Stock Solution Preparation,

 

Stock-1(10mg LAMO in 100ml methanol-100μg/ml and10mg CLO in 100ml methanol 100 μg/ml)

 

Stock-2(10mg LAMO in 50ml methanol-200μg/ml and 10mg CLO in 50ml methanol - 200 μg/ml) Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450),

 

Three replicates were made for the concentration (1, 3, 5 μg/ml of LAMO and 6, 18, 30 μg/ml of CLO) with different stock solution preparation.


 

Table 9:Robustness and Ruggedness data of LAMO and CLO*(n=3)

Conc. (μg/ml)

Lamotrigine (Mean* % RSD) (n=3)

Instrument

Stock

UV-2450

UV-1800

Stock 1

Stock 2

1

0.232 0.53

0.235 0.52

0.233 0.53

0.237 0.42

3

0.642 0.19

0.645 0.12

0.643 0.25

0.648 0.12

5

1.012 0.12

1.014 0.16

1.019 0.21

1.024 0.12

Clozapine (Mean* % RSD) (n=3)

6

0.524 0.15

0.520 0.20

0.524 0.23

0.528 0.17

18

1.444 0.08

1.446 0.11

1.446 0.22

1.447 0.14

30

2.214 0.11

2.216 0.13

2.211 0.11

2.218 0.14

 

 


Table-10: %Assay of synthetic mixture analysis (n=3)

Drugs

Result of Synthetic Mixture Analysis (n=3)

Amount of Drug

%Assay S.D.

%R.S.D.

LAMO

2 g/ml

101.970.034

0.036

CLO

12 g/ml

100.850.037

0.039

 

Application of the proposed method for analysis of EDA and ARG in synthetic mixture (ASSAY)

In that synthetic mixture (100 mg) the excipient were like Sodium Starch Glycolate, Starch, Magnesium Stearate were taken as per the required weight. With the Lamotrigine and Clozapine with the ratio (1:6) dissolved in Methanol with small volume of Solvent, Sonicate for 15 min, then make up to 100ml with methanol and filter it.

 

Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

 

Table 11: Summary of validation parameter

Sr.No

PARAMETER

Lamotrigine

Clozapine

1

Wave length Max.

308 nm

259.50 nm

2

Linearity

(g/ml) (n=6)

1-5 g/ml

6-30 g/ml

3

Regression equation

Y=0.0362x +

0.0044

0.0641x +

0.0294

4

Correlation coefficient (r2)

0.998

0.998

5

Accuracy(%Recovery) (n=3)

100.74 %

101.91%

6

Precision

Intra-day (%RSD)(n=3)

Inter-day (%RSD)(n=3)

 

0.25-0.53

0.24-0.60

 

0.16-0.70

0.11-0.32

7

LOD (g/ml) (n=10)

0.289

0.108

8

LOQ (g/ml) (n=10)

0.877

0.329

9

Robustness and

Ruggedness (%RSD)

0.12-0.53

0.08-0.23

10

Assay

101.97 %

100.85%

 

CONCLUSION:

All the parameters are validated as per ICH guidelines for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Ruggedness and Robustness of method was confirmed as no significant were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are in fair agreement.

ACKNOWLEDGEMENT:

We are sincerely thankful to Shree Dhanvantary Pharmacy College, Kim, Surat, for providing us Infrastructure facilities and moral support to carry out this research work. We are also thankful to SDPARC for giving us their special time and guidance for this research work. We also thank our colleagues for their helping hand.

 

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Received on 04.03.2015 Accepted on 01.04.2015

Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 5(2): April-June 2015; Page 79-85

DOI: 10.5958/2231-5675.2015.00013.7